Proteasome-mediated endoproteolytic cleavage initiates the processing of p100ΔCs. (A) Schematic representation of the p100 (1–665) chimeras containing p100 (1–405)-HA or GFP at their C-termini. p100 (1–665) is also represented. The filled black arrows and scissor indicate the potential initial cutting site. The black dotted arrow indicates the direction of the proteasomal processing. (B) Endoproteolytic processing of p100 (1–665) chimeras in vivo. The 293 cells were transfected with the indicated constructs, followed by IB using the indicated antibodies. The endogenous p100 is indicated as an asterisk (∗). (C) The molar ratio of the processed products from N and C termini of chimeras. The ratio of p52:p100 (1–405) generated from chimera p100 (1–665)-p100 (1–405)-HA was directly based on the densitometry quantitation of the bands detected by anti-p100N in B. The ratios of the processed p52 and GFP to the precursor p100 (1–665)-GFP were also shown.

Constitutive processing of p100ΔCs occurs at the κB promoter. (A) In vitro processing of p100 (1–665) by the 20S proteasome. In vitro-translated p100 (1–665) proteins were incubated with the purified 20S proteasome in the reaction buffer in the presence or absence of an increasing amount of the κB or Oct-1 DNA oligonucleotide for 1.5 h at 37°C, fractionated by SDS/PAGE, and visualized by autoradiography. (B) In vitro processing of p100 (1–665) by the 26S proteasome. In vitro processing assay was performed by using the 26S proteasome. (C) In vitro processing of p80HT. In vitro processing assay was performed by using in vitro-translated p80HT proteins and purified 20S proteasome. (D) Detection of p100 (1–665) in vitro processing by IB. In vitro processing reaction using cold p100 (1–665) proteins was subjected to immunoblot by using anti-p100N.

p100ΔC recruits the proteasome into the κB promoter to form a proteasome/p100ΔC/κB promoter complex. (A) Schematic representation of the Bcl-xL and IL2Rα promoters. The κB DNA sequences and transcription initial site (+1) are indicated. The primers for ChIP and their locations at the promoter are also indicated. (B) Binding of the endogenous p80HT to the Bcl-xL promoter in vitro. Nuclear extracts isolated from HuT78 cells were used for EMSA. (C) Binding of the transfected p80HT to the Bcl-xL promoter in vitro. EMSA was performed by using the nuclear extracts from 293 cells expressing C-terminally HA-tagged p80HT. (D) Association of the proteasome with the Bcl-xL promoter in HuT78 tumor cells. ChIP was performed to examine the in vivo binding of the proteasome and p80HT/p52 to the Bcl-xL promoter by using the indicated antibodies. The actin promoter was used as negative controls. (E) Recruitment of the proteasome into the κB promoter by p80HT. ChIP was performed to examine the binding of the proteasome to the Bcl-xL promoter in 293 cells expressing the indicated C-terminally HA-tagged constructs. (F) Recruitment of the proteasome to different κB promoters by different p100ΔCs. ChIP was performed to examine the binding of the proteasome to the Bcl-xL and IL2Rα promoters in FL5.12 cells stably expressing p52, LB40, or LB40 DB mutant. (G) Role of the κB DNA in the interaction between p100ΔC and the proteasome. Co-IP was performed by using in vitro-translated p80HT or p52 and purified 20S proteasome.

Amino acid D₄₁₅ is the potential endoproteolytic cleavage site required for p100ΔC constitutive processing. (A) Schematic representation of the preferential proteasomal cleavage site within p100ΔCs. The arrow indicates the endoproteolytic cleavage site. The amino acid numbers for the preferential proteasomal cleavage site are also indicated. D415, the only conserved amino acid among different species, is shown in red. (B) Essential role of the potential proteasomal cleavage site in p100ΔC constitutive processing in vivo. The 293 cells were transfected with the indicated p100 (1–665) constructs, followed by IB using anti-p100N to detect the precursor proteins and their processed product p52. (C) Essential role of the amino acid D₄₁₅ in the processing of p100ΔCs in vitro. In vitro processing assays were performed by using the indicated proteins. (D) Essential role of the amino acid D₄₁₅ in the constitutive processing of the p100ΔC chimeras. The 293 cells were transfected with the indicated chimera constructs, followed by IB using anti-p100N. (E) Effect of mutation of the proteasomal cleavage site on the nuclear localization of p100ΔCs. HeLa cells were transfected with the indicated p80HT constructs, followed by immunofluorescence assay. (F) Effect of mutation of the proteasomal cleavage site on p100ΔC binding to the Bcl-xL promoter. ChIP was performed to detect the association between p80HT and Bcl-xL promoter in 293 cells expressing C-terminally HA-tagged p80HT or its D₄₁₅A mutant. (G) Effect of mutation of the proteasomal cleavage site on the binding of p100ΔCs to the proteasome. Co-IP was performed by using purified proteasome and in vitro-translated p80HT or its S₄₁₃RDS₄₁₆ deletion mutant (labeled as Δ) in the presence or absence of the κB DNA.

The DNA-binding activity of p100ΔCs is essential for their constitutive processing. (A) Schematic representation of the N-terminal point mutations in the NF-κB2 DNA-binding domain. Amino acids R₅₂FRYGCE₅₈ within p100 or its C-terminal deletion mutants were substituted with L₅₂FLFGPV₅₈ and labeled as DB. DS, DNA-binding sequence; D, dimerization domain; N, nuclear localization sequence; ARD, ankyrin repeat domain; PID, processing inhibitory domain. The arrow indicates the processing site. LB40 and p80HT are p100 C-terminal truncation mutants that were originally identified from B cell chronic lymphocytic leukemia and T cell lymphoma, respectively. Note, p80HT is a fusion protein containing 1–665 aa of p100 at the N terminus and another three heterologous amino acids (serine-alanine-serine) at the C terminus; LB40 is equivalent to p100 (1–702). (B) κB DNA-binding activity of the endogenous p80HT. Nuclear extracts were isolated from HuT78 cells and incubated with the consensus κB DNA oligonucleotide in the presence or absence of preimmune serum (Preserum) or different NF-κB antibodies. The DNA/protein complexes are indicated. (C) κB DNA-binding activity of the transfected p100ΔCs. Nuclear extracts were isolated from 293 cells expressing C-terminally HA-tagged p80HT or p100 (1–665) and subjected to EMSA. (D) Failure of the DB mutants of p100ΔCs in binding to the κB DNA. EMSA was performed with the nuclear extracts from the indicated cells. (E) Role of the DNA-binding activity of p100ΔCs in their nuclear translocation. Immunofluorescence assays were performed by using HeLa cells transfected with the indicated constructs. (F) Role of the DNA-binding activity of p100 and its p100ΔC mutants in their processing. The 293 cells transfected with the indicated constructs were used for IB to detect p52 and its precursor proteins. The endogenous p100 is indicated as an asterisk (∗).